The increased availability and identification of genes from human and other genomes has led to an increased need for efficient expression and purification of recombinant proteins. The expression of proteins in bacteria is by far the most widely used approach for the production of cloned genes. For many reasons, expression in bacteria is preferred to expression in eukaryotic cells. For example, bacteria are much easier to grow than eukaryotic cells. More specifically, the availability of a wealth of sophisticated molecular genetic tools and thousands of mutants make E. coli, as an expression host, extremely useful for protein production. However, the high-level production of functional proteins in E. coli., especially those from eukaryotic sources has often been difficult.
IL-21 (previously designated Zalphal1 Ligand) is a member of the IL-2 family of cytokines that also includes IL-4, IL-7, IL-9, IL-13, and IL-15. Proteins in this family have been shown to have both anti-cancer and anti-viral effects. IL-21 is produced by helper T-cells, which are key regulators of immunity. Based on expression patterns of its cognate receptor and administration of the protein, it has been shown that IL-21 activates CD8+ killer T-cells and natural killer (NK) cells, two classes of lymphocytes that eradicate tumors and virally infected cells. IL-21 also stimulates select classes of B-cells. (Parrish et al., Nature 408:57-63, 2000).
Recombinant IL-21 has been produced in prokaryotic cells, in particular E. coli. The resulting bacterial produced protein is not glycosylated, and is produced in an aggregated state. Production of IL-21 from E. coli requires that the aggregated proteins be solubilized from the insoluble inclusion bodies and renatured or refolded. Without renaturation, the specific activity of the recombinant protein will be significantly reduced.
Despite advances in the expression of recombinant proteins in bacterial hosts, there exists a need for improved methods for producing biologically active and purified recombinant IL-21 proteins in prokaryotic systems which result in higher yields for protein production. These and other aspects of the invention will become evident upon reference to the following detailed description. In addition, various references are identified below and are incorporated by reference in their entirety.